Presumed common source outbreaks of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region.

Hepatitis A virus isolates from anti-HAV IgM positive sera of 70 hepatitis cases in two outbreaks and 216 other cases in Central America, 136 sporadic cases and 53 cases from an hyper-endemic region in Costa Rica, were compared by phylogenetic analyses within the VP1 region. The outbreaks in all 531 cases, in 1992 and 1999, respectively, were presumed water borne. In the first outbreak, HAV RNA could be detected in 70% of the cases sampled during 6 weeks after onset of jaundice. In the hyper-endemic region of San Ramón in Costa Rica, 1,932 cases were registered between 1972 and 1985. All isolates belonged to subtype 1A. Background isolates from Costa Rica and El Salvador tended to form separate subclusters in the phylogenetic tree construction and were mostly unrelated to subtype 1A strains from other parts of the world. Based on their amino acid sequences, four HAV strains, all related to CR326 sampled in Costa Rica in 1960, were found to have circulated in the area during the last three decades. However, on the basis of nucleotide variability the isolates from the outbreaks could be distinguished from the strains from sporadic cases and sequence analysis could confirm the epidemiological homogeneity of both outbreaks. In the hyper-endemic region, 16 different sequences were encountered forming one single subcluster. Thus, limited sequencing within the VP1 region proved useful to identify outbreaks of hepatitis A in a highly endemic area, where most strains were local and only one subtype was prevalent.

Development of low cost peptide-based anti-hepatitis C virus screening and confirmatory assays: comparison with commercially available tests.

Screening and confirmatory low cost reagent tests have been developed for detection of anti-hepatitis C virus (HCV). Assays are based on the use of specific synthetic peptides from several structural and non-structural viral proteins. The efficacy of the screening anti-HCV EIA-Spep assay was compared with both Abbott EIA 2.0 (Abbott Laboratories, North Chicago, IL) and Ortho EIA 2.0 (Ortho Diagnostic Systems, Raritan, NJ) anti-HCV detection kits and the confirmatory EIA-Cpep assay was compared with the Abbott Matrix anti-HCV confirmation test. In the EIA-Spep, a pool of 3 peptides was added to each well of a microtiter plate. In EIA-Cpep, each well was separately coated with 1 of 4 peptides and 1 recombinant protein. A total of 867 blood donor samples from Costa Rica tested simultaneously with the 3 screening assays yielded the same specificity and negative predictive values of > or =99.9% and 100%, respectively. A comparative study on voluntary blood donor samples from Honduras, Nicaragua, and El Salvador using the 2 anti-HCV confirmatory assays revealed different patterns that are 46% positive, 24% indeterminate, and 30% negative with the EIA-Cpep assay vs. 31% positive, 48% indeterminate, and 21% negative with the Matrix assay. A study of 71 patient samples from Costa Rica showed a higher correlation between initially reactive samples when analyzed by the Abbott and Ortho kits, than when the assay results were compared between the Abbott and EIA-Spep kits; the latter detected 7 and 15 non-reactive samples, respectively. These results could reflect the use of a similar antigen source for the 2 commercial assays. The presence of HCV RNA in a group of 29 samples analyzed was related to the simultaneous reactivity in all 3 screening assays. None of the discordant samples had detectable levels of HCV RNA. Economic difficulties for health care services in the developing countries of Central America have prevented implementation of routine anti-HCV blood donor screening tests. This is likely to be the primary reason for uncontrolled dissemination of HCV, and the lack of identification of potential high risk groups. Alternative low cost reagents developed locally as described in this article could be a useful tool in the control of HCV spread throughout the developing world.

Molecular epidemiology of hepatitis B virus in Central America reflected in the genetic variability of the small S gene.

The S genes of 31 Central American hepatitis B virus (HBV) strains belonging to genotypes A, C, D, and F (4, 1, 4, and 22 strains, respectively) were compared with 104 published S genes. According to the deduced S gene product, 21 genotype F strains encoded adw4, while 1 encoded ayw4. Three clusters were revealed within genotype F, which correlated with substitutions at residue 45. In a cluster of 18 Central American and 1 Alaskan strain, all had Thr45. One cluster included 2 Central American strains and 6 strains from South America and Europe, which had Leu45. Two Nicaraguan strains differed by five substitutions, including a Pro45 in the S gene product from other F strains. In conclusion, the dominating HBV genotype was F, which might be the reason for a low prevalence of HBV in the area, despite high prevalence of hepatitis A. These infections otherwise vary in parallel and are considered to reflect socioeconomic conditions.

High prevalence of GB virus C strains genetically related to strains with Asian origin in Nicaraguan hemophiliacs.

The presence of hepatitis GB virus C (GBV-C), also known as hepatitis G virus (HGV), and hepatitis C virus (HCV) were investigated in sera from 45 hemophiliacs from nine locations in Nicaragua using a nested polymerase chain reaction (PCR). Primers used to detect GBV-C and HCV derived from the helicase region and 5'UTR, respectively. Seventeen (38%) patients were positive for GBV-C RNA in serum by PCR. Twelve (27%) patients were positive for HCV RNA by PCR. Six (13%) of these were coinfected with GBV-C. Anti-HCV was detected in all the 12 HCV RNA positive hemophiliacs and in another 14 (31%) individuals, in whom GBV-C RNA was found in 2. Ten patients (22%) lacked markers for both GBV-C and HCV. The mean age of the patients positive for GBV-C but negative for HCV by PCR was significantly lower than for those negative for GBV-C but positive for HCV by PCR (P < 0.05; Student's t-test), indicating that the risk for this group of hemophiliacs to acquire GBV-C infection is higher as compared to the risk of acquiring HCV infection. Eleven GBV-C strains were sequenced in the 5'UTR. Sequence comparison to previously published GBV-C strains revealed that all 11 strains were more similar to Asian strains than to strains of European and African origin. Sequences in the NS5-B region were available for 8 HCV strains, all of which were found to belong to genotype 1a. The similarity of the Nicaraguan GBV-C strains to strains from Asia indicates that the GBV-C strains in the region presumably have an Amerindian origin. It is also considered that the HTLV II strains in the New World aboriginal populations are ancient and brought there by the ancestral Amerindian populations from Asia. Further, the genotype F of hepatitis B virus, known to represent the strains in populations with Amerindian background, predominates in Central American populations with Hispanic background. It remains to be clarified why Amerindian strains of GBV-C as well as of HBV predominate also in populations with mixed ethnic background in Central America.

Genotype F prevails in HBV infected patients of hispanic origin in Central America and may carry the precore stop mutant.

The distribution of HBV genotypes and the presence of the precore stop mutation were investigated in HBV strains from Central America. 333 HBsAg positive sera from chronic HBsAg carriers and acute hepatitis B cases from five different countries (Costa Rica, Nicaragua, Honduras, EI Salvador and Guatemala) were tested for HBV DNA by nested PCR. Genotyping by limited sequencing within the S gene was performed on 90 strains, 66 from sera with a high level of HBV DNA, and another 24 from sera positive for HBV DNA only after nested PCR. 23 of the samples were anti-HBe positive. Genotype F was found in 71 (79%), A in 13 (14%), D in 5 (6%) and C in one of the 90 sera. 18 patients with genotype F infection had anti-HBe and HBV DNA in serum. Since the three published precore sequences of genotype F strains have a C1858, which is known to prevent the precore stop mutation from G to A at position 1896, the precore and part of the core genes were sequenced from 19 anti-HBe positive sera with HBV DNA, 17 with genotype F and 2 with genotype A. The A1896 mutation was found in 11 of the 17 genotype F strains. All these had a T1858, which was also present in 5 of the 6 genotype F strains with G1896. The precore region was therefore sequenced from genotype F strains from 5 HBeAg positive sera from the five different Central American countries. These also had a T1858, which thus is the wild type substitution in genotype F in Central America. A number of mutations were recorded between residues 57 and 68 in the core protein corresponding to a unique clustering region of the genotype F strains. The predominance of genotype F in Central American populations of Hispanic origin was not anticipated since this genotype is regarded as indigenous to the Amerindian populations of the New World.

Evaluation of a pooling method for routine anti-HCV screening of blood donors to lower the cost burden on blood banks in countries under development.

A pooling system was developed for use in anti-HCV screening of voluntary blood donors at the local Central American Red Cross blood banks, in Nicaragua, El Salvador and Honduras. The commercially available second generation anti-HCV screening kit from Abbott Laboratories (North Chicago, IL) was used with a modification in the initial serum dilution procedure. Pools of five sera were selected for routine screening, based on comparative studies of individual samples and of pools with different sample sizes. During the years 1993 and 1994 a total of 89, 148 voluntary blood donors were screened and a positive prevalence rate of 0.35% was established. Of the initially positive samples, 54% confirmed positive, 30% were indeterminate and 16% were negative using the Abbott Matrix test. Significant differences of positive screening prevalence rates were found in the three countries, with average values of 0.50%, 0.23% and 0.08%, respectively, in Nicaragua, El Salvador and Honduras. These initially positive samples also showed a different confirmatory pattern with a positive rate of 64% in Nicaragua, in contrast to 20% in El Salvador. Only a few samples were available for RT-PCR amplification of HCV-RNA; however, this highly sensitive method did not appear to be more helpful than serology in confirming the HCV donor status. Overall, the data obtained indicate a fluctuation of HCV prevalence in voluntary blood donors among the three Central American countries. Further, differences were found in the percentages of initially screened positives and confirmation patterns. This information appears useful for establishing criteria in future screening policies. Thus, we suggest that the use of pooling for anti-HCV screening is beneficial in countries under development, since there are potential cost savings, as well as benefits in establishment of initial prevalence rates.

Evaluation of the specificity of an immunoprecipitin test for non-A, non-B hepatitis.

The specificity of a new antigen-antibody system (devised at the International Center for Medical Research and Training [ICMRT], San José, Costa Rica) for non-A, non-B (NANB) hepatitis was evaluated. ICMRT antigen was found in eight (21%) of 38 patients with acute NANB hepatitis; 22 patients (58%) seroconverted, including three who were positive for ICMRT antigen. Five patients with chronic NANB hepatitis were persistently positive for ICMRT antigen and negative for ICMRT antibody during several years of observation. Neither ICMRT antigen nor seroconversion was found among 11 patients with hepatitis A and 19 with hepatitis B occurring concomitantly with NANB hepatitis; only two of 56 patients with other liver diseases had ICMRT antigen, both presumably with chronic antigenemia. Seven of 128 household contacts of patients with NANB hepatitis had ICMRT antigen; 27 had antibody initially, and 35 (37% of susceptible contacts) seroconverted during the observation period. Less than 4% of household contacts of patients with hepatitis A or B seroconverted.

Hepatitis A virus infection in households.

The behavior of hepatitis A virus (HAV) infection among 980 members of 230 families in two rural districts of Costa Rica was studied prospectively from the recognition of the index case. The initial prevalence of detectable antibody (anti-HAV) ranged from 26.2% in children to 71.4% in adults. The ratio of index to household-associated infections was significantly higher among children than among adolescents and adults, indicating that children were most often responsible for the HAV introduction. The rates of household-associated cases among susceptible contacts were 70-83%; the final prevalences of anti-HAV were 90-95%. Neither index showed significant differences related to age. The ratio of clinical to silent infections in household-associated cases was uniformly 1.8:1 among children and adolescents; among adults, almost all associated infections were silent. Beginning with the 5-9-year age group, however, an immunoglobulin M response was absent in a progressively larger proportion of inapparent infections, strongly suggesting restimulation of specific immunoglobulin G antibodies by reinfection.

Immunoglobulin M level in the diagnosis of type A hepatitis.

The serum immunoglobulin M level has often but not always been found to be higher in type A than in type B hepatitis. Studies of this question, however antedated the recognition of non-A, non-B disease, and the etiologic characterization may have been mistaken in many instances. It was appropriate, therefore, to reopen the question by an investigation of 62 serologically defined cases. IgM values above 300 U/ml occurred in 28 of 33 type A episodes, three of 24 type B, and none of ten non-A, non-B cases. Although nonspecific, the IgM assay is a generally available procedure that may provide useful evidence concerning the etiologic form of acute viral hepatitis.

Identity of antibody to hepatitis B e/1 antigen as an atypical rheumatoid factor.

Chronic carriers of hepatitis B surface antigen (HBsAg) who were positive for antibody to hepatitis B e antigen were tested for precipitating specificities of the antibody and for rheumatoid factor (RF) over a two-year period. Twenty-one of 69 carriers were RF-positive by the latex-RF test with human IgG, but all were negative by the hemagglutination-RF test with rabbit IgG. Antibody to e/1 antigen was identified as an IgM antibody reactive in the latex-RF test, whereas antibody to e/2 antigen belonged to the IgG class and showed no RF reactivity. Antibody production seems to follow a sequential evolution: antibody to e/1 antigen appears early and that to e/2 antigen follows shortly thereafter. Antibody to e/2 antigen remains as the permanent antibody, whereas that to e/1 antigen sometimes disappears. The fact that antibody to e/1antigen is present only in certain chronic carriers of HBsAg would account for discrepancies in reported results of RF test in viral hepatitis.

Anti-DNA, anti-HBc correlations with RIA-detected HBeAg and anti-HBe in chronic HBsAg carriers.

The interrelations of 1) antibody to hepatitis B core antigen (HBcAg)--anti-HBc; 2) single-stranded DNA-binding antibodies (anti-DNA); and 3) the e-antigen/antibody system--hepatitis B e antigen (HBeAg) and antibody (anti-HBe), were studied in 150 hepatitis B surface antigen (HBsAg) carriers, in 43 of whom diagnostic liver biopsies had been performed. There was a good correlation between titers of anti-HBc and anti-DNA, regarded as indicators of viral and pathological activity, respectively, as well as between levels of these two antibodies and the presence of HBeAg or anti-HBE as detected by radio-immune assay (RIA). In general, HBeAg-positive carriers showed high anti-HBc and high anti-DNA titers, while the carriers positive for anti-HBe had low titers of both. These findings were in accord with the histopathological results. The three serologic parameters, anti-HBc, anti-DNA, and e-antigen/antibody, should together prove useful for the evaluation of the clinical status of chronic HBsAg carriers.

Antibodies to single stranded DNA: a diagnostic aid in chronic hepatitis B virus infections.

Serial determinations of titers of binding antibodies to single stranded DNA were performed over a period of three years in 43 type B hepatitis patients with persisting HBsAg who had either developed chronic hepatitis or were asymptomatic carriers. Patients with histopathological diagnosis of chronic active or chronic persistent hepatitis, with or without clinical symptoms, showed high titers of anti-DNA throughout the course of the disease, whereas in most of these patients the serum alanine transaminase and bilirubin levels fluctuated widely and were often normal; in such cases the elevation of anti-DNA was frequently the only positive sign present. On the other hand anti-DNA titers were within the normal range in chronic asymptomatic HBsAg carriers who showed no histopathological or biochemical changes. Anti-DNA determinations are proposed as a reliable diagnostic aid to supplement current procedures for assessment of the disease status during the course of chronic hepatitis B virus infections.

Dane particles and associated DNA-polymerase activity in saliva of chronic hepatitis B carriers.

To investigate furhter the problem of salivary transmission of type B hepatitis, salivas free of blood contamination from three HBsAg-positive carriers with chronic active hepatitis were examined by CsCl equilibrium density gradients and electron microscopy (EM). In the CsCl gradient HBsAg of whole salivas distributed in a band centered at 1.19gm/cm3 with a clearly defined shoulder at 1.24 gm/cm3; the HBsAg was found mainly in the mucous component. On EM examination, fractions from the 1.19 gm/cm3 peak contained spherical HBsAg particles of 22 +/- 3 nm diameter, whereas in the 1.24 gm/cm3 shoulder Dane particles 43 nm in diameter with 28 nm cores were found. Specific hepatitis B virus associated DNA-polymerase activity also was found in the 1.24 gm/cm3 shoulder where the Dane particles occurred and was absent from the saliva of healthy controls. When salivas were incubated for three hours at 37 degrees C the total amount of HBsAg diminished and the 1.24 gm/cm3 shoulder disappeared, probably as a result of endogenous degradation of the Dane particles and the free HBsAg. These findings clearly indicate that the hepatitis B viral particle is present in the saliva of chronic HBsAg carriers with active disease and further confirm that saliva is an important vehicle of infection.

Evidence that e-antigen is not transmitted with hepatitis B infection.

The correlation between transmission of e-antigen (e-Ag) and hepatitis B antigen (HBsAg) was investigated in primary and secondary cases of type B hepatitis occurring in 15 families. The e-Ag was not consistently transmitted in the familial environment, as were the d and y subtypes of HBsAg, but occurred in erratic fashion among genetically and epidemiologically related cases. It is concluded that e-Ag is probably the non-transmissible product of a specific individual response to hepatitis B infection.

Antibodies to single-stranded DNA: an aid in diagnosis of viral hepatitis.

In clinical and subclinical viral hepatitis a significant increase of antibodies to single-stranded DNA revealed by the prepatent stage of the disease before any elevation of serum transaminases. In type A hepatitis, a rise in anti-DNA titers was detectable one to two weeks before onset of clinical and biochemical signs; in type B hepatitis, the rise of anti-DNA coincided with or preceded the appearance of HBSAg, several weeks before the onset of clinical illness. In both hepatitis types anti-DNA titers reached a peak (640--2,560) at onset and dropped shortly after serum transaminases returned to normal at the end of acute illness. The anti-DNA response in non-A/non-B hepatitis was of similar magnitude. Anti-DNA elevation was the only positive sign found in most silent infections of either type that later showed specific seroconversion. Anti-DNA levels in noninfected contacts were in the same range as those found in a group of health individuals. The anti-DNA test is useful for early diagnosis of viral hepatitis and should be a valuable addition to current epidemiological and clinical procedures.